RBP-J dependent and independent signalling of EBNA-2 [Elektronische Ressource] / Kristina Grabušić

Grabusic, Kristina

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• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

RBP-J DEPENDENT AND INDEPENDENT
SIGNALLING OF EBNA-2







A Thesis Submitted for the Degree of Doctor of Natural Sciences

At the Faculty of Biology,

Ludwig-Maximilians-Universität München













Kristina Grabušić

thMünchen, 23 December 2003











Completed at the GSF Research Centre for Environment and Health GmbH
Institute for Clinical Molecular Biology and Tumour Genetics, München





































First Examiner: PD Dr. Bettina Kempkes


Additional Examiners: PD Dr. Ruth Brack-Werner
Prof. DrHans-Ulrich Koop ofDr. Walter Schartau

thDate of the oral examination: 10 May 2004










Mojim roditeljima

(To my parents) TABLE OF CONTENTS



1. Introduction 1
1.1. EBNA-2, an important tool of Epstein-Barr Virus 1
1.1.1. Epstein-Barr Virus 1
1.1.2. B-cells are essential for establishment and persistence of EBV infection 1
1.1.3. EBNA-2 takes part in the initial proliferating phase of
EBV life cycle in B-cells 2
1.1.4. EBV guides proliferation and differentiation of B-cells by multiple
latency programmes 3
1.1.5. In vitro EBV infection of B-cells: a model to study EBV latent genes 4
1.1.6. EBNA-2, the central regulator of transcription in latently infected B-cells 4
1.1.7. EBNA-2 associated disorders 5
1.1.8. EBV associated diseases 6
1.2 EBNA-2 signaling 7
1.2.1. EBNA-2 responsive elements 7
1.2.2. The LMP-2A promoter as a model for RBP-J dependent signalling
of EBNA-2 8
1.2.3 The LMP-1 promoter as a potential model for RBP-J independent
signalling ofEBNA-2 in B-cells 9
1.3. The EBNA-2 protein 10
1.3.1. EBNA-2 regions important for B-cell immortalisation and transactivation 10
1.3.2. EBNA-2 associated proteins that bind specifically to DNA 12
1.3.3. Further EBNA-2 associated proteins 14
1.3.4. The EBNA-2 transactivation mechanism mediated by RBP-J 14
1.3.5. The Notch signalling: the cellular pathway converging to RBP-J 15
1.4. Goal of the project 16

2. Results 18
2.1. The mutational analysis of the LMP-1 promoter 18
2.1.1. The RBP-J binding sites in the LMP-1 promoter exert repression 18
2.1.2. EBNA-2WW325FF activates the LMP-1 promoter lacking
the RBP-J binding sites 21
2.1.3. The repressive role of the RBP-J binding site versus the activating
role of the PU.1 binding site in transactivation of the LMP-1 promoter
by EBNA-2 24 2.2. Characterisation and dissection of EBNA-2 signalling by
genetic analysis 27
2.2.1. The RBP-J promoter targeting domain of EBNA-2 as a potential
multifunctional domain 28
2.2.2. The RBP-J binding region derived from Notch functionally
replaces the intrinsic RBP-J binding domain of EBNA-2
only in the activation of the LMP-2A promoter 30
2.2.3. Two EBNA-2 mutants preferentially activate either the LMP-1or
the LMP-2A promoter 33
2.2.4. EBNA-2 mutants HAdelCR7 and HACR5-9 35
2.3. Immunoprecipitation of EBNA-2 protein/DNA complexes in the
context of chromatin 38
2.3.1. Three EBNA-2 specific antibodies immunoprecipitate EBNA-2
bound to DNA in vo 38
2.3.2. EBNA-2 binds to latent viral promoters in vivo 40
2.3.3. EBNA-o the CD23 promoter vivo 42
2.4. Construction of recombinant Epstein-Barr Viruses 45
2.4.1. Introduction of a mutation into the EBNA-2 ORF of the viral genome 46
2.4.2. Establishment of stable EBV positive 293 cell lines 48
2.4.3. Production and quantification of viral supernatants 50
2.5. Infection of B-cells with recombinant EBVs 52
2.5.1. Infection of primary B-cells with recombinant EBVs and determination
of the immortalisation efficiency 52
2.5.2. LCL/CR4 growth is impaired in comparison to LCL/wtEBV 55
2.5.3. LCL/CR4 and LCL/wtEBV express comparable LMP-1 protein levels 55
2.5.4. DG75 converted with recombinant EBVs do not express LMP-1 57

3. Discussion 59
3.1. The LMP-1 promoter: EBNA-2 can induce transcription
independently of RBP-J 59
3.2. What are the possible roles of the RBP-J binding sites in
the EBNA-2 activated promoters? 62
3.3. The RBP-J targeting domain of EBNA-2 as a potential
binding site for other proteins 64
3.4. The RBP-J dependent signalling of Notch can partially
replace the RBP-J dependent signalling of EBNA-2 65
3.5. Two regions of EBNA-2 are critical for promoter targeting 67
3.6. EBNA-2 and the RBP-J signalling of EBNA-2 are absolutely
required for immortalisation of B-cells by EBV 69
3.7. The CR4 deletion in EBNA-2 strongly influences the
immortalisation efficiency of the recombinant virus and
affects the growth rate of the established LCLs 70
3.8 Outlok 72

4. Material 73
4.1. Bacterial strains 73
4.2. Cell lines 73
4.3. Material for bacterial and eucaryotic cell culture 74
4.4. Plasmids 74
4.5. Oligonucleotides 76
4.6. Antibodies 77
4.7. Probe for Southern blot analysis 77
4.8. Enzymes
4.9. Other molecular biology and chemical reagents 78
4.10. Kits and other material 78
4.11. Laboratory equipment 79

5. Methods 80
5.1. Bacterial cell culture 80
5.1.1. Maintenance and propagation of bacteria 80
5.1.2. Preparation of competent bacteria 80
5.1.3. Preparation of competent bacteria with the induced λRed system 81
5.1.4. Heat shock transformation 82
5.1.5. TSS transformation 82
5.1.6. Electroporation and homologous recombination in DH10B 83
5.1.7. Isolation of plasmid DNA 83
5.1.8. Isolation and purification of recombinant EBV DNA 84
5.2. Eucaryotic cell culture and analysis of cells 85
5.2.1. Cultivation of suspension and adherent cells 85
5.2.2. Storage of cell lines 85
5.2.3. Transient transfection by electroporation 86
5.2.4. Luciferase assay 86
5.2.5. Transfection of 293 cells and selection of stable cell clones 87
5.2.6. Production of infectious virions and quantification of viral titres 87
5.2.7. Preparation of primary B-cells 88 5.2.8. FACS analysis 88
5.2.9. Infection of primary B-cells with recombinant EBV and determination
of the immortalisation efficiency 88
5.3. Methods for the manipulation and analysis of DNA 89
5.3.1. Cloning ofrecombinant plasmids 89
5.3.2. Small scale preparation of DNA from LCL samples for PCR analysis 92
5.3.3. Polymerase chain reaction 92
5.3.4. Non-radioactive labelling of DNA fragments 93
5.3.5. Isolation of total DNA in large scale 93
5.3.6. Non-radioactive Southern blot analysis 94
5.4. Methods for the analysis of protein and protein/DNA
interactions 95
5.4.1. Preparation of protein extracts 95
5.4.2. Protein quantification 95
5.4.3. SDS-polyacrylamide gel electrophoresis 96
5.4.4. Western blotting and immunodetection of proteins 96
5.4.5. Chromatin binding and immunoprecipitation assay 97

6. Sumary 99

7. Bibliography 100
LIST OF ABBREVIATIONS



AIDS acquired immune deficiency syndrome
ATF/CRE activating transcription factor/ cAMP response element
ATP adenosine triphosphate
AUF1 AU-rich element- and poly(U)-binding and degradation factor,
identical to CBF2
BAC bacterial artificial chromosome
BALF4 BamHI A fragment leftward open reading frame number 4
BL Burkitt’s lymphoma
BZLF1 BamHI Z fragment leftward open reading frame number 1
Cam chloramphenicol
CATamacetyl transferase
cAMP cyclic adenosine monophosphate
CBF1 Cp promoter binding factor 1, identical to RBP-J
CBF2 omoter factor 2,ical to AUF1
CBP CREB-binding protein
ChIP chromatin immunoprecipitation
CIR CBF1 interacting factor
CMV cytomegalovirus
CoR co-repressor complex
CR conserved region
DEAD D-E-A-D (Asp-Glu-Ala-Asp)
dNTP 3’ deoxyribonucleoside-5’-triphosphate
DIG digoxygenin
DMSO dimethyl sulphoxide
DP103 DEAD box binding protein 103
DTT dithiothreitol
EBER Epstein-Barr virus encoded RNA
EBNA Epstein-Barr Virus nuclear antigen
EBNA-LP Epstein-Barr Viru antigen leading protein
EBV Epstein-Barr Virus
E. coli Escherichia coli
EDTA ethylene diamine tetra-acetic acid
ER oestrogen receptor
EBNA-2 REs EBNA-2 responsive elements
FACS fluorescence assisted cell sorting
FCS foetal calf serum
FITC luorescein